Synthesis of analogues of (E)-1-hydroxy-2-methylbut-2-enyl 4-diphosphate, an isoprenoid precursor and human γδ T cell activator

Article abstract of DOI:10.1021/jo701873t

(Chemical Equation Presented) (E)-1-Hydroxy-2-methyl-but-2-enyl 4-diphosphate (HMBPP) is an intermediate in the non-mevalonate pathway for the biosynthesis of isoprenoids and also serves as a very strong activator of human γδ T cells expressing Vγ9Vδ2 receptors. This paper describes the synthesis of analogues of HMBPP, in which the diphosphate group is replaced by potential isosteric moieties, i.e., carbamate, N-acyl-N′-oxy sulfamate, or aminosulfonyl carbamate functionalities. The potential of the synthesized analogues to stimulate Vγ9/Vδ2 T cell response or to inhibit GcpE and LytB, the last enzymes in the non-mevalonate pathway, was assessed.

Full text of DOI:10.1021/jo701873t

Synthesis of Analogues of (E)-1-Hydroxy-2-methylbut-2-enyl
4-Diphosphate, an Isoprenoid Precursor and Human γδ T Cell
Activator
Steven Van Hoof,† Carl Jeffrey Lacey,† Rene´ C. Ro¨hrich,‡ Jochen Wiesner,‡
Hassan Jomaa,*,‡ and Serge Van Calenbergh*,†
Laboratory for Medicinal Chemistry, Faculty of Pharmaceutical Sciences, Ghent UniVersity,
Ghent, Belgium, and Institut fu¨r Klinische Immunologie und Transfusionsmedizin,
Justus-Liebig-UniVersita¨t Giessen, Giessen, Germany
serge.Vancalenbergh@ugent.be; hassan.jomaa@uniklinikum-giessen.de
ReceiVed September 3, 2007
(E)-1-Hydroxy-2-methyl-but-2-enyl 4-diphosphate (HMBPP) is an intermediate in the non-mevalonate
pathway for the biosynthesis of isoprenoids and also serves as a very strong activator of human γδ T
cells expressing Vγ9Vδ2 receptors. This paper describes the synthesis of analogues of HMBPP, in which
the diphosphate group is replaced by potential isosteric moieties, i.e., carbamate, N-acyl-N′-oxy sulfamate,
or aminosulfonyl carbamate functionalities. The potential of the synthesized analogues to stimulate Vγ9/
Vδ2 T cell response or to inhibit GcpE and LytB, the last enzymes in the non-mevalonate pathway, was
assessed.
1. Introduction
The non-mevalonate pathway starts with the formation of
1-deoxy-D-xylulose 5-phosphate (5) by condensation of pyruvic
acid (3) and D-glyceraldehyde 3-phosphate (4) (Scheme 1). Via
2-C-methyl-D-erythritol 5-phosphate (6) the pathway leads to
(E)-1-hydroxy-2-methyl-but-2-enyl 4-diphosphate (HMBPP, 7).
This intermediate is finally transformed to IPP (1) and DMAPP
(2), in a reaction catalyzed by the ispH (or LytB) enzyme.5 This
enzyme contains an iron-sulfur cluster that suggests a radical
mechanism of transformation.6
Interestingly, HMBPP has also been identified as a very
strong activator of the human γδ T cells expressing Vγ9Vδ2
receptors.2,7 The γδ T cells constitute 0.5-5% of the human
peripheral blood T cells, the vast majority of which express the
Various disease-causing organisms such as Plasmodium
falciparum (malaria), Bacillus anthracis (anthrax), Clostridium
botulinum (botulism), and Mycobacterium tuberculosis (tuber-
culosis) use the non-mevalonate biosynthetic pathway to produce
isopentenyl diphosphate (IPP, 1) and dimethylallyl diphosphate
(DMAPP, 2).1,2 These isoprenoids serve as biosynthetic precur-
sors of a wide myriad of terpenes,3 some of them being essential
components in life cycles of any cell type. However, in
mammals IPP and DMAPP are formed exclusively via the
unrelated mevalonate pathway. Selective inhibition of the non-
mevalonate pathway would therefore be an interesting approach
to the development of new treatments for important infectious
diseases.4
(4) Pink, R.; Hudson, A.; Mourie`s, M.-A.; Bendig, M. Nat. ReV. Drug
DiscoVery 2005, 4, 727-740.
† Ghent University, Harelbekestraat 72, 9000 Gent, Belgium. Phone: +32 9
264 81 24. Fax +32 9 264 81 46.
(5) (a) Cunningham, F. X., Jr.; Lafond, T. P.; Gantt, E. J. Bacteriol.
2000, 182, 5841-5848. (b) Altincicek, B.; Duin, E. C.; Reichenberg, A.;
Hedderich, R.; Kollas, A.-K.; Hintz, M.; Wagner, S.; Wiesner, J.; Beck,
E.; Jomaa, H. FEBS Lett. 2002, 532, 437-440. (c) Rohdich, F.; Hecht, S.;
Ga¨rtner, K.; Adam, P.; Krieger, C.; Amslinger, S.; Arigoni, D.; Bacher,
A.; Eisenreich, W. Proc. Nat. Acad. Sci. U.S.A. 2002, 99, 1158-1163.
(6) Gra¨wert, T.; Kaiser, J.; Zepeck, F.; Laupitz, R.; Hecht, S.; Amslinger,
S.; Schramek, N.; Schleicher, E.; Weber, S.; Haslbeck, M.; Buchner, J.;
Rieder, C.; Arigoni, D.; Bacher, A.; Eisenreich, W.; Rohdich, F. J. Am.
Chem. Soc. 2004, 126, 12847-12855.
‡ Justus-Liebig-Universita¨t Giessen, Langhansstrasse 7, 35392 Giessen,
Germany. Phone: +49 641 99-41573. Fax +49 641 99-41589.
(1) Rohdich, F.; Kis, K.; Bacher, A.; Eisenreich, W. Curr. Opin. Chem.
Biol. 2001, 5, 535-540.
(2) Eberl, M.; Hintz, M.; Reichenberg, A.; Kollas, A.-K.; Wiesner, J.;
Jomaa, H. FEBS Lett. 2003, 544, 4-10.
(3) Eisenreich, W.; Bacher, A.; Arigoni, D.; Rohdich, F. Cell. Mol. Life
Sci. 2004, 61, 1401-1426.
10.1021/jo701873t CCC: $40.75 © 2008 American Chemical Society
Published on Web 01/23/2008
J. Org. Chem. 2008, 73, 1365-1370
1365

Van Hoof et al.
SCHEME 1. Biosynthesis of IPP (1) and DMAPP (2) via
the Non-mevalonate Pathway
These analogues, but also other Vγ9Vδ2 T cell activators such
as the bisphosphonates,15 all contain a diphosphate or phos-
phonate function. This has serious drawbacks from a therapeutic
perspective, as the charged phosphate of phosphonate group
imparts poor cellular permeability characteristics. In addition,
phosphatases can rapidly cleave the phosphate groups, resulting
in a significant or total loss of activity. This paper describes
the synthesis of new analogues of HMBPP with alternative
functionalities for the diphosphate group. These analogues
potentially constitute valuable tools to mimic or to antagonize
the HMBPP-mediated Vγ9Vδ2 T cell response or to inhibit
LytB activity. By extension, they may be valuable tool
compounds to further clarify the role of Vγ9Vδ2 T cells in
host-pathogen interactions in important diseases like tubercu-
losis.
2. Results and Discussion
Starting from the previously described TBDPS-protected
compound 8,16 several analogues were synthesized in which a
carbamate functionality replaces the diphosphate moiety of
HMBPP (Scheme 2). Having one partially positive center, this
carbamate serves as an isosteric function for a monophosphate.
The synthesis was pursued by reaction of 8 in CH2Cl2 with
different isocyanates in the presence of a catalytic amount of
triethylamine to give the corresponding carbamates 9-13 in very
good yields (84-98%). Typically, the nitrogen was substituted
with a range of aromatic groups possessing different electronic
properties as described in the Topliss tree.17 The formation of
benzylcarbamate 14 required a longer reaction time and resulted
in a lower yield of 70%. Since it was observed that removal of
the TBDPS group by using tetrabutylammonium fluoride gave
purification problems due to the polarity of the final products,
ammonium fluoride was selected as the reagent of choice. The
reaction in methanol at room temperature was slow, but reaction
rates could be improved by heating to 50 °C. This process
yielded the analogues 15-20.
Initially an N-acyl sulfamate group was selected as an
alternative isostere of the diphosphate group, since it also
contains two partially positive charged centers. Introduction of
this group can be performed with the chlorosulfonyl isocyanate
reagent. By primary addition of an appropriate alcohol, the
reagent is transformed into a carbamate-protected chlorosul-
fonamide. In our case, best results were obtained by using tert-
butanol,18 thus generating a Boc-protecting group, but other
alcohols like benzyl alcohol have been used by others in the
Vγ9Vδ2 T cell receptor. Activation and proliferation of this
subset of T cells is observed in infections with pathogens known
to possess the MEP pathway, such as tuberculosis or malaria.
Vγ9Vδ2 T cell activation appears to be important in priming
and regulating various reactions of the immune system, but its
exact function is largely unknown.8,9 Nevertheless, the evalu-
ation of natural and synthetic activators of Vγ9Vδ2 T cells as
new immunomodulatory drugs has been repeatedly suggested.10
Unlike the activation of Râ T cells by small antigenic
peptides, the Vγ9Vδ2 T cells are activated by low molecular
weight phosphorylated compounds collectively called phospho-
antigens. Specific activation of the T lymphocytes was observed
after infection with a broad range of pathogenic organisms with
use of the non-mevalonate pathway. While IPP and DMAPP
appeared to be moderate stimulators,11 HMBPP gave an
approximately 10000-fold higher response and probably rep-
resents the only phosphoantigen of physiological importance.
This stimulatory effect, however, has a high degree of structure
specificity.12 It is thus a challenge to find new immunoregulating
compounds via the syntheses of HMBPP analogues.
(13) (a) Wolff, M.; Seemann, M.; Grosdemange-Billiard, C.; Tritsch, D.;
Campos, N.; Rodr´ıguez-Concepcio´n, M.; Boronat, A.; Rohmer, M. Tetra-
hedron Lett. 2002, 43, 2555-2559. (b) Amslinger, S.; Kis, K.; Hecht, S.;
Adam, P.; Rohdich, F.; Arigoni, D.; Bacher, A.; Eisenreich, W. J. Org.
Chem. 2002, 67, 4590-4594. (c) Fox, D. T.; Poulter, C. D. J. Org. Chem.
2002, 67, 5009-5010. (d) Gao, W.; Loeser, R.; Raschke, M.; Dessoy, M.
A.; Fulhorst, M.; Alpermann, H.; Wessjohann, L. A.; Zenk, M. H. Angew.
Chem., Int. Ed. 2002, 41, 2604-2607. (e) Giner, J.-L. Tetrahedron Lett.
2002, 43, 5457-5459.
Various syntheses of HMBPP have been reported,13 but until
now only few reports on HMBPP analogues have appeared.12,14
(7) Hintz, M.; Reichenberg, A.; Altincicek, B.; Bahr, U.; Gschwind, R.
M.; Kollas, A.-K.; Beck, E.; Wiesner, J.; Eberl, M.; Jomaa, H. FEBS Lett.
2001, 509, 317-322.
(8) Vermijlen, D.; Ellis, P.; Langford, C.; Klein, A.; Engel, R.; Willimann,
K.; Jomaa, H.; Hayday, A. C.; Eberl, M. J. Immunol. 2007, 178, 4304-14.
(9) Ali, Z.; Shao, L.; Halliday, L.; Reichenberg, A.; Hintz, M.; Jomaa,
H.; Chen, Z. W. J. Immunol. 2007, 179, 8287-96.
(10) Sicard, H.; Ingoure, S.; Luciani, B.; Serraz, C.; Fournie´, J. J.;
Bonneville, M.; Tiollier, J.; Romagne´, F. J. Immunol. 2005, 175, 5471-
80.
(11) Tanaka, Y.; Morita, C. T.; Tanaka, Y.; Nieves, E.; Brenner, M. B.;
Bloom, B. R. Nature 1995, 375, 155-158.
(12) Amslinger, S.; Hecht, S.; Rohdich, F.; Eisenreich, W.; Adam, P.;
Bacher, A.; Bauer, S. Immunobiology 2007, 212, 47-55.
(14) (a) Espinosa, E.; Belmant, C.; Pont, F.; Luciani, B.; Poupot, R.;
Romagne´, F.; Brailly, H.; Bonneville, M.; Fournie´, J.-J. J. Biol. Chem. 2001,
276, 18337-18344. (b) Reichenberg, A.; Hintz, M.; Kletschek, Y.; Kuhl,
T.; Haug, C.; Engel, R.; Moll, J.; Ostrovsky, D. N.; Jomaa, H.; Eberl, M.
Bioorg. Med. Chem. Lett. 2003, 13, 1257-1260. (c) Song, Y.; Zhang, Y.;
Wang, H.; Raker, A. M.; Sanders, J. M.; Broderick, E.; Clark, A.; Morita,
C. T.; Oldfield, E. Bioorg. Med. Chem. Lett. 2004, 14, 4471-4477.
(15) Sanders, J. M.; Ghosh, S.; Chan, J. M. W.; Meints, G.; Wang, H.;
Raker, A. M.; Song, Y.; Colantino, A.; Burzynska, A.; Kafarski, P.; Morita,
C. T.; Oldfield, E. J. Med. Chem. 2004, 47, 375-384.
(16) Ikeda, S.; Shibuya, M.; Iwabuchi, Y. Chem. Commun. 2007, 504-
506.
(17) Topliss, J. G. J. Med. Chem. 1972, 15, 1006-1011.
1366 J. Org. Chem., Vol. 73, No. 4, 2008

Synthesis of Analogues of HMBPP
SCHEME 2. Synthesis of Carbamate Analogues 15-20a
a Reagents and conditions: (a) RNCO, NEt3, CH2Cl2, rt, 70-98%; (b) NH4F, MeOH, 50 °C, 88-99%.
past.19 Next the in situ-prepared chlorosulfonamide was reacted
with alcohol 8, but unfortunately the formed product 21 appeared
to be unstable. Therefore, we envisaged the synthesis of a series
of N-acyl-N′-oxy sulfamide analogues. This class of compounds
was anticipated to be more stable then the N-acyl sulfamate
group and can still serve as an isosteric moiety for the
diphosphate.
SCHEME 3. Synthesis of N-Acyl-N′-oxy Sulfamate
Analogues 30-35a
Thus, alcohol 8 was transformed into a hydroxylamine via a
Mitsunobu reaction by using N-hydroxy phthalimide as the acid
in the presence of diisopropylazodicarboxylate and triph-
enylphosphine. This reaction rendered product 22 in a good
yield. Deprotection of the phthaloyl group with hydrazine
hydrate in ethanol proceeded smoothly to give hydroxylamine
23 in 94% yield. This product was treated with in situ-prepared
N-Boc-chlorosulfonamide as described above to give the N-Boc-
N′-oxy product 24. In contrast to compound 21, product 24 is
very stable. Removal of the Boc group was realized by the
careful addition of a solution of trifluoroacetic acid in dichlo-
romethane at 0 °C and maintaining the resulting solution at
0 °C for 24 h.20
For the acylation of compound 25, literature precedence
supported the use of acid chlorides in the presence of triethy-
lamine and 4-dimethylaminopyridine,21 but in our case these
conditions did not render any product at all. Another literature
procedure, the direct coupling of the carboxylic acid and the
amine with a carbodiimide reagent,22 was also completely
ineffective. When using carboxylic acids activated as the
N-hydroxysuccinimide ester and DBU as the base,23 fairly good
results could be obtained. The best results, however, were
generated by performing a transamidation reaction, using
thiazolidinethiones as the activated form of the chosen acids24
in the presence of DBU. To our knowledge this reagent has
never been used before for the acylation of sulfamates. This
approach gave compounds 26-30 in fair to good yields. Finally,
the products 31-35 were formed by the removal of the
protecting group with use of ammonium fluoride, giving high
yields (80-97%), except for the acetyl derivative (43%). The
a Reagents and conditions: (a) DIAD, PPh3, N-hydroxyphthalimide, THF,
5 °C to rt, 4.5 h, 85%; (b) H2NNH2‚H2O, THF, EtOH, rt, 4 h, 94%; (c)
ClSO2NHBoc, CH2Cl2, pyridine, 0 °C, 84%; (d) TFA, CH2Cl2, 0 °C, 20 h,
81%; (e) appropriate N-acylthiazolidinethione, DBU, THF, rt, 40-81%;
(f) NH4F, MeOH, rt, 43-97%.
intermediate 24 was deprotected by the same method to yield
product 36 in moderate yield (63%) (Scheme 3).
An unusual reverse approach delivers the possibility of
coupling two entities via an aminosulfonyl carbamate group that
represents an isosteric group of the diphosphate moiety.25
Chlorosulfonyl isocyanate first reacts with the alcohol 8 in
toluene at 0 °C to form the carbamate function. The resulting
chlorosulfonyl carbamate could serve as the electrophile for a
substitution with any amine, but since this intermediate is very
unstable and easily hydrolyses, these amines should be very
pure. The impractical use of, for example, liquid ammonia could
be circumvented by the addition of 2.2 equiv of pyridine. The
Burgess-type salt that is formed is stable to water, so even
aqueous solutions of an amine can be used to perform the
substitution reaction.26 This was exemplified by the use of
aqueous ammonia in a one-pot reaction to give compound 37
(18) (a) Raga¨ına, Z.; Abdaoui, M.; Aouf, N.-E.; Dewynter, G.; Montero,
J.-L. Tetrahedron 2000, 56, 381-387. (b) Zhong, J.; Lai, Z.; Groutas, C.
S.; Wong, T.; Gan, X.; Alliston, K. R.; Eichhorn, D.; Hoidal, J. R.; Groutas,
W. C. Bioorg. Med. Chem. 2004, 12, 6249-6254. (c) Salim, S. S.;
Bellingham, R. K.; Brown, R. C. D. Eur. J. Org. Chem. 2004, 800-806.
(19) Park, J. D.; Kim, D. H.; Kim, S.-J.; Woo, J.-R.; Ryu, S. E. J. Med.
Chem. 2002, 45, 5295-5302.
(20) Dewynter, G.; Aouf, N.; Criton, M.; Montero, J.-L. Tetrahedron
1993, 49, 65-76.
(21) Hamprecht, G.; Puhl, M.; Reinhard, R.; Sagasser, I.; Schmidt, T.;
Goetz, N.; Zierke, T. PCT Int. Appl. WO 2003097589, 2003.
(22) Lee, J.; Kim, S. E.; Lee, J. Y.; Kim, S. Y.; Kang, S. U.; Seo, S. H.;
Chun, M. W.; Kang, T.; Choi, S. Y.; Kim, H. O. Bioorg. Med. Chem. Lett.
2003, 13, 1087-1092.
(25) (a) Camarasa, M. J.; Fernandez-Resa, P.; Garcia-Lopez, M. T.; De
las Heras, F. G.; Mendez-Castrillon, P. P.; Alarcon, B.; Carrasco, L. J. Med.
Chem. 1985, 28, 40-46. (b) Castro, A.; Erickson, S. E.; Shechter, I.;
Spencer, T. A. Bioorg. Chem. 1996, 24, 242-250. (c) Nguyen, C.;
Kasinathan, G.; Leal-Cortijo, I.; Musso-Buendia, A.; Kaiser, M.; Brun, R.;
Ruiz-Pe´rez, L. M.; Johansson, N. G.; Gonza´lez-Pacanowska, D.; Gilbert,
I. H. J. Med. Chem. 2005, 48, 5942-5954.
(23) Somu, R. V.; Boshoff, H.; Qiao, C.; Bennett, E. M.; Barry, C. E.;
Aldrich, C. C. J. Med. Chem. 2006, 49, 31-34.
(24) Nagao, Y.; Seno, K.; Kawabuta, K.; Miyasaka, T.; Takao, S.; Fujita,
E. Tetrahedron Lett. 1980, 21, 841.
J. Org. Chem, Vol. 73, No. 4, 2008 1367

Van Hoof et al.
SCHEME 4. Synthesis of Aminosulfonyl Carbamate Analogues 41-44a
a Reagents and conditions: (a) ClSO2NCO, toluene, 0 °C, 30 min; (b) pyridine, toluene, 0 °C, 30 min; (c) RNH2, 0 °C to rt, 76-97%; (d) NH4F, MeOH,
rt, 14-92%.
TABLE 1. Inhibition of GcpE and LytB Activity by the Target
in 97% yield. Other amines that were used are methylamine,
Compounds at 1 mM
benzylamine, and O-TBDPS-protected hydroxylamine. Depro-
% residual activity at 1 mM
tection of the hydroxyl function(s) by using ammonium fluoride
compd
GcpE
LytB
in methanol at room temperature finally yielded compounds 41-
44 (Scheme 4). The yield for hydroxylamine analogue 44 was
significantly lower (14%) than that for the others (88-92%), a
result that may be explained by stability issues.
15
16
17
18
19
20
31
32
33
34
35
36
98
96
94
90
81
89
94
66
95
95
99
99
90
85
89
62
95
92
79
87
98
98
102
61
All final compounds were tested in the Vγ9Vδ2 T cell
activation assay in parallel with HMBPP as positive control.
As expected, significant outgrowth of Vγ9Vδ2 T cells was
observed with HMBPP at concentrations down to 0.1 nM.
However, there was no activity with the test compounds up to
100 µM. This result supports previous findings that the
diphosphate group of HMBPP is essential for potent Vγ9Vδ2
T cell activation. It was demonstrated that only very minor
modifications of this group such as in (E)-4-hydroxy-3-methyl-
but-2-enylmethylenediphosphonate (HMB-PCP), which repre-
sents the bis-phosphonate analogue of HMBPP, result in 10000-
fold reduced activity in the Vγ9Vδ2 T cell activation assay.27
A QSAR study by Gossman and Oldfield revealed that four
essential components arranged in the appropriate relative
geometry are critical for γδ T cell activation: an H-bond donor
(e.g., the OH group of HMBPP (7)), a hydrophobic feature (e.g.,
the methyl group of HMBPP), and two negative ionizable groups
(e.g., the two pyrophosphate phosphate groups of HMBPP).28
While all analogues investigated contain the former two
pharmacophore features, our data suggest that the polar diphos-
phate mimetics investigated are unable to bioisosterically replace
the pyrophosphate moiety of HMBPP, despite the fact that a
NH group flanked by a carbonyl and a sulfonamide as in 31-
36 and 41-44 will be deprotonated at physiological pH.
The potential of compounds 15-20 and 31-36 to inhibit
GcpE and LytB was also assessed (Table 1). At 1 mM
concentration, carbamate 18 and analogues 32 and 36, both
featuring an N-acyl-N′-oxy sulfamate moiety, were found to
marginally inhibit GcpE or LytB.
phate isosteres. The N-acyl-N′-oxy sulfamate function was
formed via coupling of the isoprenoid alcohol and an in situ-
protected chlorosulfamide, followed by deprotection and acy-
lation with thiazolidinethione reagents. The aminosulfonyl
carbamate moiety was prepared via a three-step one-pot reaction,
with a Burgess-type salt as an intermediate. The isoprenoid part
of HMBPP was left unchanged.
All compounds investigated failed to show significant bio-
logical activity.
3. Experimental Section
General Procedure for the Synthesis of Compounds 9-14.
To a 0.1 M solution of compound 8 in dry dichloromethane were
added the appropriate isocyanate (1.1 equiv) and triethylamine (0.1
equiv). The reaction mixture was stirred at room temperature until
completion was determined via TLC analyses. The reaction was
quenched by the addition of methanol, and the mixture was
concentrated under reduced pressure. The resulting residue was
purified by column chromatography to yield compounds 9-14.
(2E)-4-(tert-Butyldiphenylsilyloxy)-3-methylbut-2-en-1-yl Phe-
nylcarbamate (9). Preparation of the title compound according to
the general procedure described above gave 127 mg of colorless
In conclusion, we have reported the synthesis of three types
of HMBPP analogues. In one type of compounds, a carbamate
function mimicking a phosphate group was incorporated. In the
two other groups, N-acyl-N′-oxy sulfamate and aminosulfonyl
carbamate functions, respectively, were introduced as diphos-
1
oil (90%). Rf 0.23 (hexane/ethyl acetate 9/1); H NMR (300.01
MHz, acetone-d6) δ 8.65 (1H, br s), 7.74-7.69 (4H, m), 7.60-
7.57 (2H, m), 7.49-7.39 (6H, m), 7.33-7.26 (2H, m), 7.01 (1H,
ddt, J ) 7.6, 7.0, 1.2 Hz), 5.81 (1H, tqt, app t sext, J ) 7.0, 1.5
Hz), 4.73 (2H, dq, app br dd, J ) 7.0, 0.6 Hz), 4.15 (2H, q, J )
0.6 Hz), 1.72 (3H, m), 1.06 (9H, s); 13C NMR (75.00 MHz, acetone-
d6) δ 153.8 (C), 139.8 (C), 139.7 (C), 135.6 (CH), 133.6 (C), 130.1
(CH), 128.9 (CH), 128.0 (CH), 122.7 (CH), 118.5 (CH), 67.9 (CH2),
60.8 (CH2), 26.5 (CH3), 19.2 (C), 13.0 (CH3); exact mass (ESI-
MS) calculated for C28H33NO3Si [M + Na+] 482.2128, found
482.2121.
(26) Masui, Y.; Watanabe, H.; Masui, T. Tetrahedron Lett. 2004, 45,
1853-1856.
(27) Reichenberg, A.; Hintz, M.; Kletschek, Y.; Kuhl, T.; Haug, C.;
Engel, R.; Moll, J.; Ostrovsky, D. N.; Jomaa, H.; Eberl, M. Bioorg. Med.
Chem. Lett. 2003, 13, 1257-60.
(28) Gossman, W.; Oldfield, E. J. Med. Chem. 2002, 45, 4868-4874.
1368 J. Org. Chem., Vol. 73, No. 4, 2008

Synthesis of Analogues of HMBPP
General Procedure for the Synthesis of Compounds 15-20.
To a 0.2 M solution of the carbamates 9-14 in methanol was added
ammonium fluoride. The reaction mixture was heated to 50 °C and
stirred as such until complete reaction was visible by TLC analyses.
Concentrating under reduced pressure gave a residue that was
purified by column chromatography to afford the desired unpro-
tected carbamates 15-20.
(2E)-4-Hydroxy-3-methylbut-2-en-1-yl Phenylcarbamate (15).
Preparation of the title compound according to the general procedure
described above gave 47 mg of colorless oil (94%). Rf 0.23 (hexane/
ethyl acetate 1/1); 1H NMR (300.01 MHz, acetone-d6) δ 8.60 (1H,
br s), 7.58-7.54 (2H, m), 7.32-7.25 (2H, m), 7.03-6.98 (1H, m),
5.65 (1H, tqt, app t sext, J ) 7.0, 1.5 Hz), 4.68 (2H, br dd, J )
7.0, 0.6 Hz), 3.97-3.90 (3H, m); 13C NMR (75.00 MHz, acetone-
d6) δ 153.8 (C), 141.5 (C), 139.7 (C), 128.9 (CH), 122.7 (CH),
118.4 (CH), 118.0 (CH), 66.5 (CH2), 60.9 (CH2), 13.1 (CH3); exact
mass (ESI-MS) calculated for C12H15NO3 [M + Na+] 244.0950,
found 244.0942.
(300.01 MHz, acetone-d6) δ 10.08 (1H, br s), 8.86 (1H, br s), 7.74-
7.69 (4H, m), 7.50-7.40 (6H, m), 5.78 (1H, tqt, app t sext, J )
6.9, 1.5 Hz), 4.57 (2H, dd, J ) 6.9, 0.6 Hz), 4.15 (2H, m), 1.68
(3H, m), 1.47 (9H, s), 1.07 (9H, s); exact mass (ESI-MS) calculated
for C26H38N2O6SSi [M + Na+] 557.2117, found 557.2106.
N-{[(2E)-4-(tert-Butyldiphenylsilyloxy)-3-methylbut-2-en-1-yl]-
oxy}sulfamide (25). To a solution of 24 (2.760 g, 5.16 mmol) in
dry dichloromethane (10.3 mL) was added at 0 °C a 50% solution
of trifluoroacetic acid in dichloromethane (9 mL) in a dropwise
manner over 10 h. The reaction was then stirred at 0 °C for another
10 h. The reaction mixture was concentrated and the remaining
residue was coevaporated with dichloromethane (20 mL) two times.
Purification by column chromatography (dichloromethane/ethanol
98/2) gave pure 25 (1.806 g, 81%) as an oil. Rf 0.24 (dichlo-
romethane/ethanol 98/2); 1H NMR (300.01 MHz, acetone-d6) δ 8.42
(1H, br s), 7.73-7.70 (4H, m), 7.47-7.40 (6H, m), 6.31 (2H, m),
5.79 (1J, br t, J ) 6.9 Hz), 4.55 (2H, d, J ) 6.9 Hz), 4.14 (2H, s),
1.67 (3H, s), 1.07 (9H, s); 13C NMR (75.00 MHz, acetone-d6) δ
140.9 (C), 136.2 (CH), 134.3 (C), 130.7 (CH), 128.6 (CH), 119.2
(CH), 72.7 (CH2), 68.8 (CH2), 27.2 (CH3), 19.8 (C), 13.9 (CH3);
exact mass (ESI-MS) calculated for C21H30N2O4SSi [M + H+]
435.1773, found 435.1770.
General Procedure for the Synthesis of Compounds 26-29.
To a 0.2 M solution of 25 in dry THF were added the appropriate
thiazolidinethione reagent (1 equiv) and DBU (1 equiv). The
reaction was stirred at room temperature until completion. Tetram-
ethylguanidine (0.2 equiv) was added and stirring was continued
for 1 h. Next the reaction mixture was diluted with dichloromethane
and washed with 1 N solution of HCl and brine. Drying over
MgSO4, filtration, and concentration under reduced pressure gave
a residue that was purified by column chromatography to give the
respective compounds 26-29.
N-[({[(2E)-4-(tert-Butyldiphenylsilyloxy)-3-methylbut-2-en-1-
yl]oxy}amino)sulfonyl]benzamide (26). Preparation of the title
compound according to the general procedure described above gave
100 mg of a colorless oil (62%). 1H NMR (300.01 MHz, acetone-
d6) δ 11.00 (1H, br s), 9.03 (1H, s), 8.06-8.03 (2H, m), 7.71-
7.65 (5H, m), 7.57-7.52 (2H, m), 7.45-7.36 (6H, m), 5.76 (1H,
tqt, app t sext, J ) 6.9, 1.5 Hz), 4.58 (2H, dd, J ) 6.9, 0.6 Hz),
4.13 (2H, m), 1.67 (3H, m), 1.05 (9H, s); 13C NMR (75.00 MHz,
acetone-d6) δ 166.7 (C), 142.2 (C), 136.3 (CH), 134.3 (C), 132.5
(C), 130.7 (CH), 129.7 (CH), 129.2 (CH), 128.7 (CH), 118.2 (CH),
73.4 (CH2), 68.8 (CH2), 27.2 (CH3), 19.8 (C), 13.9 (CH3); exact
mass (ESI-MS) calculated for C28H34N2O5SSi [M + K+] 577.1594,
found 577.1582.
N-{[(2E)-4-(tert-Butyldiphenylsilyloxy)-3-methylbut-2-en-1-yl]-
oxy}phtalimide (22). Compound 8 (5.00 g, 14.68 mmol), triph-
enylphosphine (4.62 g, 17.62 mmol, 1.2 equiv), and N-hydroxyph-
thalimide (2.87 g, 17.62 mmol, 1.2 equiv) were dissolved in dry
THF (58.72 mL) and the solution was cooled to 5 °C. Diisopropyl
azodicarboxylate (3.47 mL, 17.62 mmol, 1.2 equiv) was added
dropwise. The reaction was stirred below 10 °C for 3 h, and then
allowed to warm up to room temperature. After 1.5 h the reaction
mixture was concentrated under reduced pressure. The resulting
residue was purified via column chromatography (hexane/ethyl
1
acetate 8/2) to give pure 22 (6.07 g, 85%) as an oil. H NMR
(300.01 MHz, DMSO-d6) δ 7.87 (4H, m), 7.61-7.58 (4H, m),
7.47-7.39 (6H, m), 5.87 (1H, tqt, app t sext, J ) 7.5, 1.5 Hz),
4.80 (2H, d, J ) 7.8 Hz), 4.05 (2H, s), 1.60 (3H, s), 0.98 (9H, s);
13C NMR (75.00 MHz, DMSO-d6) δ 164.3 (C), 144.4 (C), 135.9
(CH), 135.8 (CH), 133.7 (C), 130.9 (CH), 129.4 (C), 128.9 (CH),
124.2 (CH), 116.3 (CH), 73.6 (CH2), 67.9 (CH2), 27.5 (CH3), 19.7
(C), 14.3 (CH3); exact mass (ESI-MS) calculated for C29H31NO4Si
[M + K+] 524.1659, found 524.1655.
(2E)-4-(Aminooxy)-1-(tert-butyldiphenylsilyloxy)-2-methyl-
but-2-ene (23). To a solution of 22 (11.14 g, 22.94 mmol) in THF
(57.35 mL) and ethanol (57.35 mL) was added hydrazine hydrate
(3.345 mL, 68.82 mmol). The reaction mixture was stirred at room
temperature for 4 h and then diluted with ether (500 mL) and
washed with water (2 × 200 mL) and brine (150 mL). Drying over
Na2SO4, filtration, and concentration under reduced pressure gave
a residue that was purified by column chromatography (dichlo-
romethane/methanol 99/1) to yield pure 23 (7.64 g, 94%) as an
oil. 1H NMR (300.01 MHz, DMSO-d6) δ 7.65-7.60 (4H, m), 7.50-
7.40 (6H, m), 5.89 (2H, br s), 5.65 (1H, tqt, app t sext, J ) 6.6,
1.5 Hz), 4.12 (2H, dd, J ) 6.6, 0.9 Hz), 4.07 (2H, s), 1.58 (3H,
m), 1.01 (9H, s); 13C NMR (75.00 MHz, DMSO-d6) δ 137.4 (C),
134.8 (CH), 132.9 (C), 129.8 (CH), 127.8 (CH), 119.4 (CH), 71.0
(CH2), 67.5 (CH2), 26.5 (CH3), 18.8 (C), 13.4 (CH3); exact mass
(ESI-MS) calculated for C21H29NO2Si [M + H+] 356.2045, found
356.2036.
N-[({[(2E)-4-(tert-Butyldiphenylsilyloxy)-3-methylbut-2-en-1-
yl]oxy}amino)sulfonyl]cyclohexane Carboxamide (30). Prepara-
tion of the title compound according to the general procedure
described above gave 186 mg of white crystals (81%). Mp 133-
1
134 °C; H NMR (300.01 MHz, acetone-d6) δ 10.43 (1H, br s),
8.75 (1H, s), 7.73-7.69 (4H, m), 7.48-7.40 (6H, m), 5.77 (1H,
tqt, app t sext, J ) 6.9, 1.5 Hz), 4.56 (2H, dd, J ) 6.9, 0.6 Hz),
4.14 (2H, br d, J ) 0.9 Hz), 2.42 (1H, tt, J ) 11.2, 3.2 Hz), 1.90-
1.85 (2H, m), 1.78-1.72 (2H, m), 1.67 (3H, m), 1.65-1.60 (1H,
m), 1.50-1.19 (5H, m), 1.07 (9H, s); 13C NMR (75.00 MHz,
acetone-d6) δ 175.7 (C), 141.9 (C), 136.3 (CH), 134.3 (C), 130.7
(CH), 128.7 (CH), 118.2 (CH), 73.3 (CH2), 68.7 (CH2), 45.3 (CH),
29.7 (CH2), 27.2 (CH3), 26.3 (CH2), 26.0 (CH2), 19.9 (C), 13.9
(CH3); exact mass (ESI-MS) calculated for C28H40N2O5SSi [M +
Na+] 567.2325, found 567.2319.
tert-Butyl [({[(2E)-4-(tert-Butyldiphenylsilyloxy)-3-methylbut-
2-en-1-yl]oxy}amino)sulfonyl]carbamate (24). To a solution of
chlorosulfonyl isocyanate (1.13 mL, 12.95 mmol, 5 equiv) in dry
dichloromethane (20 mL), cooled to 0 °C, was added dropwise a
solution of tert-butanol (1.30 mL, 13.73 mmol, 5.3 equiv) in dry
dichloromethane (4 mL). The mixture was stirred at 0 °C for 1.5 h
and then added dropwise to a solution of 23 (920 mg, 2.59 mmol)
in dry pyridine (10.36 mL), cooled to 0 °C. The reaction mixture
was allowed to warm up to room temperature slowly, and stirred
for 17 h. Reaction workup was performed by diluting with ethyl
acetate (150 mL) then washing with 5% citric acid solution (2 ×
150 mL), water (150 mL) ,and brine (150 mL). Drying over MgSO4,
filtration, and concentration under reduced pressure gave a residue
that was purified by column chromatography (dichloromethane/
General Procedure for the Synthesis of Compounds 31-36.
To a 0.2 M solution of compounds 26-30 in methanol was added
ammonium fluoride (2 equiv). The mixture was stirred at room
temperature until the reaction was complete as indicated by TLC
analysis. Removal of solvent under reduced pressure gave a residue
that was purified by column chromatography, to yield compounds
31-36.
N-[({[(2E)-4-Hydroxy-3-methylbut-2-en-1-yl]oxy}amino)-
sulfonyl]benzamide (31). Preparation of the title compound
1
ethanol 99/1) to yield pure 24 (1.16 g, 84%) as an oil. H NMR
J. Org. Chem, Vol. 73, No. 4, 2008 1369

Van Hoof et al.
according to general procedure described above gave 91 mg of a
gum (91%). 1H NMR (300.01 MHz, acetone-d6) δ 8.95 (1H, br s),
8.06-8.02 (2H, m), 7.67-7.61 (1H, m), 7.55-7.48 (2H, m), 5.59
(1H, tqt, app t sext, J ) 6.9, 1.5 Hz), 4.53 (2H, dd, J ) 6.9, 0.6
Hz), 3.93 (2H, s), 1.65 (3H, m); 13C NMR (75.00 MHz, acetone-
d6) δ 143.5 (C), 134.0 (CH), 133.2 (C), 129.5 (CH), 129.3 (CH),
117.9 (CH), 73.5 (CH2), 67.2 (CH2), 13.9 (CH3); exact mass (ESI-
MS) calculated for C12H16N2O5S [M + Na+] 323.0678, found
323.0669.
General Procedure for the Synthesis of Compounds 37-40.
To a 0.75 M solution of 8 in dry toluene, cooled to 0 °C, was
added dropwise chlorosulfonyl isocyanate (1 equiv). After stirring
for 30 min, the reaction was triturated to a concentration of 0.1 M
by the addition of dry toluene. Pyridine (2.2 equiv) was added and
stirring was continued for 30 min at 0 °C. Subsequently, a solution
of the appropriate amine (6 equiv) in water or THF was added.
The reaction was stirred at 0 °C for 2 h and then at room
temperature until completion was noted. The reaction mixture was
poured into a mixture of water and EtOAc. The pH was lowered
to 1 by adding concentrated sulfuric acid. The aqueous layer was
extracted with EtOAc and the combined organic layers were washed
with brine. After drying over MgSO4, filtering, and concentrating
the solution, a residue was obtained that was purified by column
chromatography to yield compounds 37-40.
(Coy Laboratory Products, Inc., Grass Lake, USA) floated with a
gas mixture consisting of 95% N2 and 5% H2. Residual O2 was
removed with palladium catalysts. Buffers were degassed in an
ultrasound bath by bubbling a stream of helium through the liquid.
Before use, the buffers were equilibrated overnight in the tent under
stirring. For the enzyme activity assays a spectrophotometer (DU
530 with a Peltier temperature control module, Beckman Coulter)
was installed inside the tent. The activity of GcpE and LytB was
determined by monitoring the oxidation of dithionite-reduced methyl
viologen at 732 nm (ꢀ732 ) 2200 M-1 cm-1). For the GcpE assay,
the reaction mixture consisted of 150 mM NaCl, 30 mM Tris-HCl
(pH 7.5), 0.2% bovine serum albumin (BSA), 2 mM methyl
viologen, 1 mM MEcPP, and 2.5 µM GcpE in a total volume of
0.8 mL. For the LytB assay, the reaction mixture consisted of 150
mM NaCl, 30 mM Tris-HCl (pH 7.5), 0.2% BSA, 2 mM methyl
viologen, 1 mM HMBPP, and 0.1 µM LytB in a total volume of
0.8 mL. The methyl viologen was partly reduced by the addition
of sodium dithionite until an extinction between 1.3 and 1.4 at 732
nm was reached, corresponding to 0.59 to 0.64 mM reduced methyl
viologen. Typically, 25 µL of a 10 mM sodium dithionite stock
solution was added to the reaction mixture. The solid sodium
dithionite was stored in the oxygen-free tent and the stock solution
freshly prepared under anaerobic conditions. For the inhibition
assays, the test compounds were dissolved in DMSO at 100 mM
and added to the reaction mixture at a final concentration of 1 mM.
The activity was recorded in comparison to a mock control with
DMSO.
(2E)-4-(tert-Butyldiphenylsilyloxy)-3-methylbut-2-en-1-yl (Ami-
nosulfonyl)carbamate (37). Preparation of the title compound
according to the general procedure described above gave 136 mg
of white crystals (97%). Rf 0.22 (hexane/ethyl acetate 65/35); mp
Vγ9Vδ2 T Cell Activation Assay. Flow cytometric analysis of
human Vγ9Vδ2 T cells was basically performed as described.31
A
1
78-79 °C; H NMR (300.01 MHz, acetone-d6) δ 9.92 (1H, br s),
total of 2 × 105 peripheral blood mononuclear cells (PBMC) were
seeded in 200 µL of RPMI 1640 medium supplemented with 25
mM HEPES, 2 mM L-glutamine, 25 µg/mL of gentamycin, 10
U/mL recombinant human interleukin-2, and 10% human AB
serum. The test samples were dissolved in DMSO at 10 mM and
added to the assay in 3-fold serial dilutions ranging from 0.14 to
100 µM. As negative control, DMSO was used at the same dilutions.
As positive control, HMBPP in a 10-fold serial dilution ranging
form 0.01 nM and 10 µM was used. All compounds were tested in
duplicate with PBMCs from three different donors. After incubation
for 6 days at 37 °C and 5% CO2, the cells were analyzed on an
Epics XL flow cytometer supported by Expo32 software (Beckman
Coulter), using CD3-FITC and TCRVgamma9-PC5 antibodies
(Beckman Coulter).
7.73-7.69 (4H, m), 7.50-7.41 (6H, m), 6.66 (2H, br s), 5.78 (1H,
tqt, t sext, J ) 7.0, 1.5 Hz), 4.75 (2H, dd, J ) 7.0, 0.6 Hz), 4.15
(2H, br s), 3.41 (3H, m), 1.06 (9H, s); 13C NMR (75.00 MHz,
acetone-d6) δ 152.3 (C), 140.8 (C), 135.6 (CH), 133.5 (C), 130.1
(CH), 128.1 (CH), 117.6 (CH), 67.9 (CH2), 62.1 (CH2), 26.6 (CH3),
19.2 (C), 13.1 (CH3); exact mass (ESI-MS) calculated for
C22H30N2O5SSi [M + Na+] 485.1543, found 485.1537.
General Procedure for the Synthesis of Compounds 41-44.
To a 0.2 M solution of compounds 36-40 in methanol was added
ammonium fluoride. The reaction was stirred at room temperature
until completion was determined by TLC analysis. Removal of the
solvent under reduced pressure gave a residue that was purified by
column chromatography to yield compounds 41-44.
(2E)-4-Hydroxy-3-methylbut-2-en-1-yl (Aminosulfonyl)car-
bamate (41). Preparation of the title compound according to the
general procedure described above gave 35 mg as a sticky oil (92%).
Acknowledgment. This study was supported by grants from
the European Commission (STREP, LSH-2003-2.3.0-1) and
INTAS (03-51-4077) and by the Deutsche Forschungsgemein-
schaft Grant JO565/1-1 (to H. J.).
1
Rf 0.15 (dichloromethane/methanol 9/1); H NMR (300.01 MHz,
acetone-d6) δ 11.08 (1H, br s), 7.39 (2H, br s), 5.53 (1H, tqt, app
t sext, J ) 7.3, 1.5 Hz), 4.90 (1H, t, J ) 5.6 Hz), 4.64 (2H, d, J )
7.0 Hz), 3.82 (2H, br d, J ) 5.3 Hz), 1.62 (3H, br s); 13C NMR
(75.00 MHz, acetone-d6) δ 152.4 (C), 142.4 (C), 117.0 (CH), 66.3
(CH2), 62.1 (CH2), 13.1 (CH3); exact mass (ESI-MS) calculated
for C6H12N2O5S [M + Na+] 247.0365, found 247.0359.
GcpE and LytB Inhibition Assay. Recombinant GcpE and LytB
of Thermus thermophilus and Aquifex aeolicus, respectively, were
produced as described.29,30 Protein purification and conduct of the
enzyme assays was carried out under anaerobic conditions in a tent
Supporting Information Available: General experimental
procedures, analytical data on compounds 10-14, 16-20, 27-29,
32-36, 38-40, and 42-44, 13C NMR spectra of compounds 9-20,
22-23, and 25-44, and 1H NMR spectrum of compound 24. This
material is available free of charge via the Internet at http://pubs.
acs.org.
JO701873T
(30) Altincicek, B.; Duin, E. C.; Reichenberg, A.; Hedderich, R.; Kollas,
A. K.; Hintz, M.; Wagner, S.; Wiesner, J.; Beck, E.; Jomaa, H. FEBS Lett.
2002, 532, 437-40.
(31) Constant, P.; Davodeau, F.; Peyrat, M.A.; Poquet, Y.; Puzo, G.;
Bonneville, M.; Fournie´, J. J. Science 1994, 264, 267-70.
(29) Kollas, A. K.; Duin, E. C.; Eberl, M.; Altincicek, B.; Hintz, M.;
Reichenberg, A.; Henschker, D.; Henne, A.; Steinbrecher, I.; Ostrovsky,
D. N.; Hedderich, R.; Beck, E.; Jomaa, H.; Wiesner, J. FEBS Lett. 2002,
532, 432-436.
1370 J. Org. Chem., Vol. 73, No. 4, 2008